LECTURE 22: AXONAL GUIDANCE

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LECTURE 22 AXONAL GUIDANCE
REQUIRED READING Kandel text, Chapter 54
ESTABLISHMENT OF NEURONAL IDENTITY AXONAL
GUIDANCE TO TARGETS NEUROTROPHIC SUPPORT FROM
TARGET TISSUES SYNAPTOGENESIS
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HOW IS PROPER WIRING OF NERVOUS SYSTEM ACHIEVED?
THEORY 1 AXON PROJECTIONS ARE IMPRECISEPROPER
CONNECTIONS ARE WORKED OUT BY
ELECTRICAL ACTIVITIES DURING EARLY
EXPERIENCE THEORY 2 CHEMOSPECIFICITY
MODEL.AXONS ARE DIRECTED TO THEIR PRECISE
TARGETS BY CHEMICAL SIGNALS
THAT EXIST INDEPENDENT OF EXPERIENCE
Roger Sperrys retinal axon regeneration
experiment supported the chemospecificity
model When optic nerve is cut, axons regenerate
and restore normal sight and behavior When optic
nerve is cut and the eyeball is rotated 180
deg in socket, axons regenerate and restore
sight, but behavior is maladaptive FROG THINKS
UP IS DOWN And LEFT is RIGHT
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AXON GUIDANCE IS A MULTI-STEP PROCESS

1. Migration towards retinal exit point (axon
   convergence)
2. Turning to exit retina and fasiculation
3. Fasiculated extension towards chiasm
4. Ipsilateral vs. contralateral migration at optic
   chiasm (divergence point)
5. Fasiculated extension into brain
6. Projection into lateral geniculate or into optic
   tectum (divergence point)
7. Projection a certain distance along surface of
   tectum (divergence step)
8. Diving into the tectum
9. Stopping at specific depths into tectum
   (divergence step)
10. Synaptogenesis

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AXON DIVERGENCES AT OPTIC CHIASM ALLOW
CONVERGENCE OF CONGRUENT INFORMATION FROM BOTH
RETINAS
Nasal hemiretinal axons cross over at
chiasm (contralateral projection) Temporal
hemiretinal axons do not cross over (ipsilateral
projection)
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ACTIN CYTOSKELETON DYNAMICS IN GROWTH CONE DURING
ATTRACTION AND REPULSION GROWTH CONE STRUCTURE
ACTIN FILAMENTS IN FILOPODIA AND LAMELLIPODIA
MICROTUBULES ALONG AXON AND IN CORE OF GROWTH CONE
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ACTIN CYTOSKELETON DYNAMICS IN GROWTH CONE DURING
ATTRACTION AND REPULSION LAMELLIPODIA/FILOPODIA
DRIVE AXON EXTENSION
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ACTIN CYTOSKELETON DYNAMICS IN GROWTH CONE DURING
ATTRACTION AND REPULSION REPULSIVE CUES CAUSE
GROWTH CONE COLLAPSE
GROWTH CONE BEFORE SEMAPHORIN EXPOSURE SAM
E GROWTH CONE AFTER SEMAPHORIN EXPOSURE
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ACTIN CYTOSKELETON DYNAMICS THE ROLE OF
RHO-FAMILY G PROTEINS
Gas, Gai, Gaq
45,000 MW
HETEROTRIMERIC
H-Ras, K-Ras, N-Ras
21,000 MW
RAS FAMILY
SMALL GTPases
RhoA, Rac, Cdc42
21,000 MW
RHO FAMILY
RalA, RalB
21,000 MW
RAL FAMILY
GDP
GTP
ACTIVE
INACTIVE
G GTP
G GDP
Pi
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ACTIN CYTOSKELETON DYNAMICS THE ROLE OF
RHO-FAMILY G PROTEINS
Common structure of small G proteins allows
design of constitutively active (CA) and
dominant negative (DN) mutant proteins CA-RhoA ,
CA-Rac , CA-Cdc42 expressed in fibroblasts induce
specific architectural structures
FOCAL ADHESIONS STRESS FIBERS
LAMELLIPODIA
FILOPODIA
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ACTIN CYTOSKELETON DYNAMICS THE ROLE OF
RHO-FAMILY G PROTEINS
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EXTRACELLULAR AXON GUIDANCE MOLECULES ACT THROUGH
RHO-FAMILY G PROTEINS TO MODIFY GROWTH CONE
ARCHITECTURE AND STEER DIRECTION OF OUTGROWTH
ATTRACTANT GUIDANCE MOLECULES LOCAL
ACTIVATION OF RAC and CDC42 LOCAL EXTENSION
OF FILOPODIA and SPREAD OF LAMELLIPEDIA GROWT
H CONE EXTENDS TOWARD ATTRACTANT
REPULSIVE GUIDANCE MOLECULES LOCAL
ACTIVATION OF RHO LOCAL ASSEMBLY OF
ACTOMYOSIN BRIDGES GROWTH CONE CONTRACTS
AWAY FROM REPELLANT
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DIFFERENT TYPES OF AXON GUIDANCE CUES
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TOPOGRAPHIC VISUAL MAP PROJECTED ONTO TECTUM
THE SURFACE OF TECTUM AS A TOPOLOGICALLY
PRESERVED REPRESENTATION OF THE VISUAL
FIELD AXONS FROM MOST ANTERIOR OF RETINA PROJECT
TO MOST POSTERIOR TECTUM AND VISA VERSA WHAT IS
NATURE OF THE GUIDANCE SYSTEM?
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AN AXONAL REPELLENT PRODUCED BY TECTUM
POSTERIOR RETINA AXONS WONT GROW ALONG POSTERIOR
TECTAL MEMBRANES THEY GROW ALONG ANTERIOR TECTAL
MEMBRANES OR NO MEMBRANES ANTERIOR RETINA
AXONS SHOW NO SUBSTRATE PREFERENCE CONCLUSION PO
STERIOR TECTUM PRODUCES A CHEMOREPELLENT
SPECIFIC FOR POSTERIOR RETINA AXONS THE
CHEMOREPELLENT WAS PURIFIED FROM TECTUM TISSUE
HOMOGENATES REPELLENT FOUND TO BE PROTEIN NOW
TERMED EPHRIN
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EPHRINS AND THEIR RECEPTORS
EPHRINS ARE CELL-SURFACE PROTEINS A-TYPE ARE
GPI-ANCHORED B-TYPE ARE TRANSMEMBRANE EPHRIN
RECEPTORS ARE RECEPTOR TYROSINE KINASES THE
SIGNALING CAPACITIES OF EPHRIN RECEPTORS ARE VERY
DIFFERENT FROM OTHER RTKs, SUCH AS
THE NEUROTROPHIN RECEPTORS
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GRADIENT EXPRESSION OF EPHRINS ON TECTUM AND
RECEPTORS ON RETINAL AXONS
EPHRIN EXPRESSION IS HIGHEST ON POSTERIOR
TECTUM EPH EXPRESSION IS HIGHEST ON POSTERIOR
RETINAL GANGLION CELLS AND THEIR AXONS, WHICH
STOP IN ANTERIOR TECTUM LOW EPH EXPRESSION ON
ANTERIOR GANGLION CELLS ALLOWS THEIR AXONS TO
ADVANCE TO POSTERIOR TECTUM
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ECTOPIC EPH EXPRESSION DIVERTS POSTERIOR RETINAL
AXONS
PATCHES OF ECTOPIC EPHRIN EXPRESSION WAS ACHIEVED
BY INFECTING TECTUM WITH REPLICATION-COMPETENT
VIRAL VECTOR EXPRESSING EPH POSTERIOR RETINAL
AXONS WITH HIGH EPHRIN RECEPTOR LEVELS AVOID
EPHRIN OVEREXPRESSION PATCHES ANTERIOR RETINAL
AXONS ARE IMMUNE TO EPHRIN PATCHES (A MUTATION
ELIMINATING EPHRIN EXPRESSION IN BRAIN
ALLOWS POSTERIOR RETINAL AXONS TO PROJECT
THROUGHOUT TECTUM)
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EPHRIN INDUCES EPH RECEPTOR CLUSTERING AND
ACTIVATES THE RECEPTOR CYTOPLASMIC DOMAIN TO
INITIATE SIGNALING LEADING TO REPULSION
EPHRIN
EPH
SIGNALING REPULSION
NGF
TRK-EPH
SIGNALING REPULSION
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CHEMOATTRACTANT PRODUCED BY FLOOR PLATE FOR
COMMISURAL AXONS
COMMISURAL AXONS PROJECT VENTRALLY TO FLOOR
PLATE FLOOR PLATE EXPLANT ATTRACT COMMISURAL
AXONS IN EXPLANT OF DORSAL SPINAL CORD
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IDENTITY OF FLOOR PLATE CHEMOATTRACTANT AND ITS
RECEPTOR
COMMISURAL AXONS IN NETRIN AND DCC MUTANT MICE
FAIL TO PROJECT TO FLOOR PLATE
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NETRIN/DCC INTERACTION CAN ALSO BE
CHEMOREPULSIVE ROLE OF CYCLIC AMP?
UNC-5 STATUS DETERMINES WHETHER DCC-POSITIVE AXON
IS ATTRACTED OR REPELLED BY NETRIN
TROCHLEAR MOTONEURONS NORMALLY REPELLED BY
NETRIN, BUT ARTIFICIAL ACTIVATION OF PKA IN CELLS
CAUSES ATTRACTION TO NETRIN SOURCE
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CONSERVED FAMILIES OF GUIDANCE MOLECULES AND
THEIR RECEPTORS
n
GUIDANCE MOLECULES
GUIDANCE RECEPTORS
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AXON NAVIGATION THROUGH MULTIPLE GUIDANCE REGIONS
REQUIRES CHANGES IN GROWTH CONE SENSITIVITY TO
INDIVIDUAL GUIDANCE CUES
SENSITIVITY TO ATTRACTANT IS LOST!!!
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MECHANISMS FOR CHANGES IN SENSITIVITY TO AXON
GUIDANCE CUES I. CHANGE IN INTRACELLULAR CYCLIC
NUCLEOTIDE LEVELS
Early in their outgrowth, retinal axons travel
into netrin-expressing region of optic
stalk Netrin is attractive guidance cue Axons
express high cAMP
Later in their outgrowth, retinal axons avoid
netrin-expressing deep regions of thalamus and
tectum Netrin is repulsive guidance cue Axons
express low cAMP
Netrin mRNA (blue) Retinal ganglion axons
(brown)
from Shewan et al., Nature Neuroscience 5955
(2002)
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MECHANISMS FOR CHANGES IN SENSITIVITY TO AXON
GUIDANCE CUES II. SILENCING A GUIDANCE CUE

• COMMISURAL AXONS ARE
• ATTRACTED TO MIDLINE FLOOR PLATE,
• CROSS ONCE,
• AND NEVER CROSS AGAIN
• Midline floor plate produces
• Netrin attractant and Slit repellent
• Initially, axons are attracted by netrin and
• are insensitive to slit repulsion
• Upon reaching floor plate, Slit does two things
• SILENCES netrin attraction
• INDUCES Slit repulsion
• SLIT ACTS LOCALLY TO MODIFY
• THE PROPERTIES OF THE
• GROWTH CONE

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SLIT SILENCES NETRIN BY GENERATING ROBO/DCC
RECEPTOR COMPLEXES AND SILENCING MUST PRECEDE
SLIT-MEDIATED REPULSION
Slit induces DCC/Robo complexes, and the
cytoplasmic domain of Robo binds the cytoplasmic
domain of DCC to block attractive signaling The
formation of DCC/Robo complexes may also promote
exocytosis of Robo-containing vesicles, thereby
increasing surface Robo and allowing for
Slit-mediated repulsion
Only after axon passes through floor plate, it is
no longer sensitive to floor plate
attraction Only after axon passes through
floor plate, it becomes sensitive to Slit
repulsion